This grant proposal is aimed at the molecular dissection of the process of transcriptional activation from enhancer sites in eukaryotes. Transcription is a fundamental process that controls cell growth and cellular differentiation. Further, the mechanism of activation by many activators is conserved in eukaryotes that range from yeast to mammals. The design of the experiments is to isolate adaptor molecules that connect transcriptional activators bound at enhancers to general transcription factors bound at the TATA box. We have already isolated two candidates for such molecules - - ADA2 and ADA3. Next, we aim to demonstrate the protein- protein contacts that connect the activators to the ADA molecules, the ADA molecules to each other, and the ADA complex to the general transcriptional machinery. Further, we aim to clarify the role of the general transcription factor, TFIIB, in activation. Binding of TFIIB to activators and to RNA polymerase II will be studied. We will also employ in our binding assays TFIIB mutants that display altered activation properties in vivo. We aim to build a molecular picture that displays all of the relevant proteins that connect activators to the general factors and cause activation of transcription. We further hope to delineate those surfaces that participate in these protein-protein interactions.